These studies are a continuation of experiments originated in our laboratory, from a previously funded RO1. Since there are no good biomarkers or preventative strategies, infection-associated pregnancy complications represent an important clinical problem. The regulation of Toll-like receptor (TLR) expression and function at the maternal-fetal interface may determine whether a pregnancy succeeds or fails. By understanding the mechanisms that control how trophoblast TLRs function, only then can we move to clinical applications to determine better ways to predict pregnancy outcome and treat women at risk of infection-associated pregnancy complications. Excessive placental apoptosis has been associated with preeclampsia and preterm labor; however the initial trigger and mechanisms involved are not fully understood. We propose that infections represent a potential trigger for placental apoptosis, and that some TLRs can mediate this response. Specifically, our central hypothesis is that some bacterial and viral components, through TLRs, induce trophoblast apoptosis, leading to adverse pregnancy outcome, such as preterm labor or preeclampsia. In our published studies we found TLR2 activation by gram-positive bacterial peptidoglycan (PDG), and TLR8 activation by viral ssRNA, trigger human first trimester trophoblast apoptosis via two distinct pathways: TLR2 directly mediates apoptosis, and this is differentially regulated by the TLR2 co-receptor, TLR6. In contrast, TLR8 indirectly mediates apoptosis by upregulating the cell's production of IFN2. Our objectives are to further characterize the cellular and molecular mechanisms by which TLR2 and TLR8 mediate trophoblast apoptosis in response to bacterial and viral components, and to determine their impact on pregnancy outcome. Within this overall goal, we will address innovative areas in which there are major gaps in our knowledge, such as the: regulation of TLR expression by DNA methylation; regulation of trophoblast miRs by TLRs; function of TLR8 in the trophoblast; and the effect of viral ssRNA on pregnancy outcome. We will also apply translate our in vitro findings into a study to develop better predictive and preventative strategies. Thus, our specific aims are to: 1. Determine the regulation of TLR2-induced apoptosis in response to bacterial components. 2. Determine the mechanism by which TLR8 functions in the trophoblast in response to viral components. 3. Determine the role of microRNAs in the regulation of TLR2- and TLR8-mediated trophoblast apoptosis. 4. Evaluate the role of TLRs in pregnancy. While the link between bacterial infections and pregnancy complications is well established, less is known about how viral infections affect pregnancy. Despite a recent increase in our understanding of the role of placental TLRs in pregnancy, our knowledge about the specific mechanisms involved is still limited, as is the role of TLRs in mediating apoptosis. Our findings will further our understanding of normal placental function, and will lead to a better understanding, prediction and treatment of infection-associated pregnancy complications. PUBLIC HEALTH RELEVANCE: The major objective of this proposal is to understand the mechanisms by which bacterial and viral infections, through the Toll-like receptors, induce placental apoptosis. Our studies will advance our understanding of the pathogenesis of infection-associated pregnancy complications, such as preterm labor. Our findings may also lead to new diagnostic markers, therapeutic strategies, and clues for novel drug targets.